Habitat Segregation Patterns of Container Breeding Mosquitos: The Role of Urban Heat Islands, Vegetation Cover, and Income Disparity in Cemeteries of New Orleans

Aedes aegypti and Aedes albopictus are important pathogen-carrying vectors that broadly exhibit similar habitat suitability, but that differ at fine spatial scales in terms of competitive advantage and tolerance to urban driven environmental parameters. This study evaluated how spatial and temporal patterns drive the assemblages of these competing species in cemeteries of New Orleans, LA, applying indicators of climatic variability, vegetation, and heat that may drive habitat selection at multiple scales. We found that Ae. aegypti was well predicted by urban heat islands (UHI) at the cemetery scale and by canopy cover directly above the cemetery vase. As predicted, UHI positively correlate to Ae. aegypti, but contrary to predictions, Ae. aegypti, was more often found under the canopy of trees in high heat cemeteries. Ae. albopictus was most often found in low heat cemeteries, but this relationship was not statistically significant, and their overall abundances in the city were lower than Ae. aegypti. Culex quinquefasciatus, another important disease vector, was also an abundant mosquito species during the sampling year, but we found that it was temporally segregated from Aedes species, showing a negative association to the climatic variables of maximum and minimum temperature, and these factors positively correlated to its more direct competitor Ae. albopictus. These findings help us understand the mechanism by which these three important vectors segregate both spatially and temporally across the city. Our study found that UHI at the cemetery scale was highly predictive of Ae. aegypti and strongly correlated to income level, with low-income cemeteries having higher UHI levels. Therefore, the effect of excessive heat, and the proliferation of the highly competent mosquito vector, Ae. aegypti, may represent an unequal disease burden for low-income neighborhoods of New Orleans that should be explored further. Our study highlights the importance of considering socioeconomic aspects as indirectly shaping spatial segregation dynamics of urban mosquito species.


Introduction
The container-breeding mosquitos, Aedes aegypti and Aedes albopictus, are important pathogen-carrying vectors that thrive in urban environments. Although these mosquitos may share habitat suitability broadly, at fine spatial scales they have different levels of tolerance to urban driven environmental changes and differing competitive advantages [1,2]. Aedes albopictus is thought to be a superior competitor due to its ability to better exploit food resources, such as leaf litter within container habitats at the larval stage [1][2][3]. This competitive advantage has been linked to a displacement of Ae. aegypti, from urban areas across the United States, where Ae. albopictus has been introduced [2]. Aedes aegypti's competitive inferiority may be alleviated by its higher ability to tolerate heat and egg desiccation [1,2], which may be especially common within cities due to urban heat island (UHI) effects. This interplay between competitive ability to exploit resources and the ability in the winter to 28.5 • C in the summer. New Orleans is one of the few locations in the USA, where Ae. aegypti, and Ae. albopictus are known to currently coexist [2]. dition, we sampled two cemeteries that had been reported to have populations of Ae. aegypti, on previous studies [18]. The seven cemeteries selected were in the planning districts of Lakeview (Metairie and Holt cemeteries, New Orleans, LA, USA), Uptown (Carrolton cemetery, New Orleans, LA, USA), Central City (Lafayette no. 1 and Lafayette no. 2 cemeteries, New Orleans, LA, USA) and St. Roch (St. Roch and St. Vincent de Paul cemeteries, New Orleans, LA, USA). These cemeteries span a gradient of income levels above and below the median income for New Orleans (Table 1). Sampling was limited to areas of the cemetery within nearest to residential neighborhoods (~150 m) to ensure feeding sources for adult female mosquitos within their flight range.  Within New Orleans, we focused our study in cemeteries across the city. Cemeteries are ideal locations for the study of these species. They provide ample containers as larval rearing sites, as well as shelter and food sources for adults, both in terms of sugar sources from ornamental plants, and blood sources from cemetery visitors [17]. For research, they are ideal locations to study as they provide easier access to larvae samples, compared to sampling in private residences [17], and unlike discarded container habitats (e.g., trash and waste) they are often distributed equally across neighborhoods of different socioeconomic characteristics. Because vegetation was an important factor of interest, we first selected five cemeteries that had a mixture of trees and open spaces for comparison. In addition, we sampled two cemeteries that had been reported to have populations of Ae. aegypti, on previous studies [18]. The seven cemeteries selected were in the planning districts of Lakeview (Metairie and Holt cemeteries, New Orleans, LA, USA), Uptown (Carrolton cemetery, New Orleans, LA, USA), Central City (Lafayette no. 1 and Lafayette no. 2 cemeteries, New Orleans, LA, USA) and St. Roch (St. Roch and St. Vincent de Paul cemeteries, New Orleans, LA, USA). These cemeteries span a gradient of income levels above and below the median income for New Orleans (Table 1). Sampling was limited to areas of the cemetery within nearest to residential neighborhoods (~150 m) to ensure feeding sources for adult female mosquitos within their flight range.

Mosquito Larvae Collection
Sampling took place monthly between January 2020 and December 2020, with the exception of March 2020 due to COVID 19 stay at home orders. Two people inspected cemetery vases until larvae positive vases were found, both under the canopy of tree and under open space. If no larvae sample was found after 30 min. of searching a cemetery, the search concluded. Because of the size of the cemetery, this timeframe was considered sufficient to cover the entire cemetery by two people. The largest cemetery, Metairie, was sampled in the area closest to a residential neighborhood, and divided into three sections, all of which were inspected for a maximum of 30 min. On each sampling occasion, a maximum of six samples were collected in this larger cemetery, whereas a maximum of two samples (one open, one closed) were collected on other cemeteries. Water in the container, including larvae was mixed and collected entirely for analysis in the laboratory. Samples were stored in a cooler until larvae identification. Larvae were counted and identified to species using morphological keys [19]. During each sampling event, the sampled vase was emptied of contents and returned to its place. Therefore, some vases may have been sampled in more than one occasion. However, we considered each sample independent because contents were entirely removed between sampling events.
During the months of May to December 2020, a container survey was conducted on each sampling event, where all water-filled containers found with and without larvae during the 30-min search were counted and classified as larvae positive or larvae negative. This effort was meant to capture the number of vases inspected during our search period and the actual availability of larvae habitat (i.e., water-filled vases) on each of the cemeteries. We calculated a container index value or the percentage (%) of water holding containers with larvae [20] for these sampling months (Table 1). While our larvae sampling was destructive, we only limited our collection to one open and one closed canopy vase. Therefore, even if we found more than one larvae positive container, we did not collect more than two samples per cemetery per occasion to limit our interference with larvae habitat in subsequent sampling events.

Temporal Abiotic Factors
We characterized temperature and rainfall variability through the sampling year using Daymet: Daily Surface Weather Data, which provides data on a 1-km grid for North America [21]. Based on the sampling date for each month, we estimated rainfall and temperature in 1-4-week time lags. Climatic variables were estimated for each sampling date and for each cemetery, individually.

Cemetery Scale Habitat Characterization
We delimited a 200 m buffer around each cemetery using ArcGIS (ver. 10.8) to demark the flight range (~200 m) of Aedes species [22]. Within this buffer, we characterized UHI, vegetation cover, and median income. We used the UHI layer developed by the Trust for Public Lands [23]. This layer consists of a 30 m grid for cities in the U.S., depicting a heat severity index, ranging in values from 1 to 5, with 1 being slightly above the mean for the city and 5 being a significantly above the mean for the city [23]. The index was developed following the Jenks Natural Breaks classification method [24], and derived from Landsat 8 imagery band 10 (ground level thermal sensor) from summers 2018 and 2019. For vegetation cover, we used the USDA Forest Service percent tree canopy cover layer, a 30 m raster layer, calculated using a random forest regression algorithm and based on the National Agriculture Imagery Program (NAIP) aerial photographs [25,26]. Median income was determined using the American Community Survey 2017-5-year summary files [27] datasets.

Container Scale Habitat Characterization
For each larvae positive container sampled, we documented water temperature with a handheld thermometer, and estimated canopy cover with a spherical convex densitometer (Forestry Suppliers, Inc. Jackson, MS, USA). All vases consisted of approximately the same water volume (1 L or less), containers larger than this volume were rare, and were not considered, in order to standardize for this variable.

Statistical Analysis
The influence of temporal abiotic factors (temperature and rainfall variables with time lags) on larvae abundance per species were examined using quasipoisson regressions to account for over dispersion. Prior to the analyses, all variables were mean centered and scaled, except for the larvae response variables, which were treated as counts.
The relative importance of factors at the cemetery scale and the container scale was examined using a generalized linear mixed effects model (GLMM), using a negative binomial distribution to account for over dispersion. For the container scale characterization, samples were classified in terms of canopy cover above the container as either open (<60% canopy cover) or closed (>60% canopy cover), and transformed into a dummy variable (1: closed, 0: open).
The evaluated variables at the cemetery scale (200 m buffer), heat index, vegetation index and income, were converted into categories of high and low (1: high, 0: low) following the gradient illustrated in Figure 2 and Table 1. These three variables were highly correlated to each other, according to Pearson correlation coefficients of 0.90+ (Appendix A) and thus were examined separately. For the GLMMs, we added the variable cemetery as a random effects factor, to control for the influence of sampling on the same site on repeated occasions. The response variable for all models consisted on counts for each of the species.

General Trends
We found a total of 87 larvae positive containers across all sampled cemeteries, 52 All analyses were performed in R version 3.6.1 [28]. Quasipoisson regressions were conducted using the stats package in R. For the GLMM we used package lme4 [29]. We tested for overdispersion using package blmeco [30]. Some of our graphs were developed using the package ggplot2 [31].

General Trends
We found a total of 87 larvae positive containers across all sampled cemeteries, 52 under an open canopy, and 35 under a closed canopy ( Table 2). From these vases, we collected a total of 1611 mosquito larvae. The most abundant species was Cx. quinquefasciatus (n = 748), followed by Ae. aegypti (n = 559), and lastly Ae. albopictus (n = 304). We found more Cx. quinquefasciatus in open canopy (n = 535) than in closed canopy vases (n = 213). We found more Ae.  Table 1). Two cemeteries (St. Roch, Holt) could not be accessed in certain months, and therefore we did not include their data for the container index calculations. Coexistence among the species of interest was detected in 8 out of 87 vases samples (~9%) (Appendix B). The vases that had coexistence were located in Carrolton, Holt, Metairie, and St. Vincent de Paul cemeteries. For these vases, we observed a trend for a greater proportion of Ae. albopictus larvae in closed canopies, while the opposite was true for Ae. aegypti. This trend was not statistically tested, as there were so few of cases (four open canopy vases, four closed canopy vases). Coexistence in vases occurred only during peak mosquito season (May-October), and most often detected in the St. Vincent de Paul cemetery.

Temporal Abiotic Factors
Overall, temperature ranged from a minimum of 7.59 • C in December, to a maximum of 33.53 • C in August. Accumulated rainfall on a 4-week lag ranged from a minimum of 40 mm in October to a maximum of 345 mm in August. Detailed rainfall and temperature estimates for each sampling occasion are included in Appendix C, and a subset of these values are illustrated on Figure 3.

Temporal Abiotic Factors
Overall, temperature ranged from a minimum of 7.59 °C in December, to a maximum of 33.53 °C in August. Accumulated rainfall on a 4-week lag ranged from a minimum of 40 mm in October to a maximum of 345 mm in August. Detailed rainfall and temperature estimates for each sampling occasion are included in Appendix C, and a subset of these values are illustrated on Figure 3.  (Table 3).   (Table 3). No significant relationships were found between Ae. aegypti and the climatic factors (Table 3). Aedes albopictus varied proportionally with maximum temperature with a 4-week time lag (Pr(>|t|) = 0.046), and to minimum temperature with 2-4-week time lags, (Pr(>|t|)<0.05, Table 3). No relationship was found between Ae. albopictus and rainfall moving averages. For Cx. quinquefasciatus a significant negative relationship was found with maximum temperature with 4-week time lag (Pr(>|t|) = 0.029). This species showed a negative relationship between minimum temperatures with 2-4 week time lags (Pr(>|t|< 0.05, Table 3). A negative relationship was found between Cx. quinquefasciatus and rainfall at the 1-week lag (Pr(>|t|) = 0.022) and the 4-week lag (Pr(>|t|) = 0.040).
Because Cx. quinquefasciatus numbers declined during the peak mosquito season, starting in May, and were reduced to zero by August (Figure 3), we considered this species to be temporally segregated from the other two species in the locations under study. Therefore, we excluded this species from subsequent analyses, as these focus on evaluating the spatial segregation patterns between species coinciding temporally in the sampled locations.

Cemetery Scale vs. Container Scale Factors
For Ae. aegypti, there was a significant effect of both the container canopy cover and heat index at the cemetery scale ( Figure 4, Table 4). Ae. aegypti counts were positively associated to UHI (Pr(>|z| = 0.001, Table 4), and positively associated to canopy cover (Pr(>|z| = 0.0007, Table 4). Therefore, Ae. aegypti was most closely associated to vases within high heat cemeteries, and with closed canopy cover ( Figure 4, Table 4). No other significant association was found between Ae. aegypti and the other cemetery scale variables measured.   While Ae. albopictus was more frequently collected in low heat cemeteries (Figure 4), there was no statistically significant relationship between UHI and this species (Table 4). There was no clear trend between canopy cover and Ae. albopictus, and no statistically significant relationship between the other cemetery scale variables (vegetation index, median income) and the species.
Random effects were examined using 97.5% confidence intervals. Confidence interval values did not overlap zero on any of the models tested, including cemetery and canopy scale for both species. The confidence intervals for random effects on the statistically significant models are as follows: Ae. aegypti/heat index (0.3346-1.5716); Ae. aegypti/canopy cover (0.9408-4.0412).

Discussion
Our study found that Ae. aegypti is a more abundant species than Ae. albopictus in the area sampled within the city of New Orleans (Figure 1). Within the cemeteries sampled, Ae. aegypti was most closely associated to locations with a high heat index at the cemetery scale (UHI). While the results were not statistically significant for Ae. albopictus, its distribution pattern shows the opposite trend, as the species was most often found in low heat cemeteries. These results suggest a spatial segregation pattern driven by heat tolerance of Ae. aegypti, as proposed in our hypotheses. These findings also support previous studies that classify Ae. aegypti as the more urban species, and Ae. albopictus as more associated to vegetated suburban/rural areas in Florida, USA [2,32,33], as well as several tropical cities [2,32,[34][35][36].
We observed in our study that heat was negatively correlated to vegetation cover, and negatively correlated to median income (Appendix A). This suggests a disparity in terms of vegetation cover and heat exposure between low and high-income neighborhoods in New Orleans. More studies are needed to test this trend as we only sampled seven locations across the city. However, these findings are not surprising, as previous studies have reported unequal exposure to heat in low-income neighborhoods consistently across many cities of the USA [37,38]. Higher UHI exposure has been consistently shown to be linked to mortality and morbidity in humans [11], while access to green spaces may promote a variety of physical and mental health benefits [12]. Therefore, it would be important to further evaluate these apparent disparities to heat exposure and vegetation cover across socioeconomic divides in New Orleans.
Our findings show that high heat in particular exacerbates exposure to Ae. aegypti, which, according to previous studies, is a more effective disease vector than Ae. albopictus [14,15]. If we layer this finding with that of other studies suggesting how urban disrepair and abandonment create habitat for mosquitos and disproportionally affect low-income neighborhoods [5][6][7][8], we can see how there are multiple complex pathways by which poverty can be potentially linked to higher mosquito borne disease risk in our cities. Future studies should determine the role of mosquito habitat segregation in driving differences in relative disease risk within a city, accounting for differences in abundance and vector competence of the mosquito species present. Because of the tight correlation, we found that with UHI and income level in this study, we were not able to disentangle temperature related drivers, to container habitat availability drivers. The interaction between container habitat availability and UHI could present interesting dynamics for mosquito dispersal and therefore could be the basis for future research.
Contrary to our prediction, canopy cover at the container scale was not predictive of the presence of Ae. albopictus. Moreover, contrary to our prediction, canopy cover was positively associated with Ae. aegypti, in the locations were this species resided, which were high heat cemeteries, with low vegetation cover at the cemetery scale. Although Ae. aegypti tolerates higher temperatures, it makes sense that it would prefer to be under shaded conditions in these high heat localities, to experience less desiccation risk, and less extreme heat. Due to the lack of significant association found between Ae. albopictus to vegetation cover at any scale, our study does not support the hypothesis of habitat selection by this species is driven by the presence of trees and its competitive superiority at exploiting leaf litter. In laboratory studies, Ae. albopictus shows a superior ability to exploit leaf litter [39,40], and a preference to lay eggs in water infused with leaf detritus over water only containers [41]. However, there is no evidence in the literature that under natural conditions the species would preferentially select this food source, as it may be able to use other food sources available such as grass clippings and animal carcasses, which may offer more nutrient availability [39,40], and are not contingent on the presence of vegetation, or direct canopy cover.
The fact that Ae. albopictus was lower in abundance than Ae. aegypti in the sampled locations (which were all highly urbanized), and that the species was most often found in low heat cemeteries, suggests that temperature may be an important determinant of this species presence/absence across different parts of the city of New Orleans. Forecasting models have used the optimal temperature ranges for Ae. aegypti (21.3-34.0 • C) and Ae. albopictus (19.9-29.4 • C) to predict a decline in Ae. albopictus in the tropics in response to projected temperature increases due climate change [42]. It is possible that similar to climate driven temperature shifts, microscale variation in temperature ranges due UHI may also lead to temperature limits across a city's landscape, shaping the segregation patterns of these two competing species.
Previous studies support the role of heat at limiting habitat suitability for Ae. albopictus within cities. A study conducted across urban to rural gradient in Athens, GA found that adult abundance of the species increased with temperature until a threshold of about 30 • C, after which adult abundance declined [43]. A similar study in Athens GA found that temperature was higher and relative humidity lower in urban vs. rural/suburban areas of the city, and that these microclimate differences resulted in lower Ae. albopictus larvae survival in the urban sites [44]. In Sao Paulo, where both species coexist, temperature increases have favored a greater expansion of Ae. aegypti across the city, relative to Ae. albopictus [45]. Accordingly, dengue infections have been positively correlated to UHI in Sao Paulo [46], while modelling studies using thermal optima for disease transmission suggest a higher thermal optimum for transmission of dengue by Ae. aegypti (29.1 (28.4-29.8)) than Ae. albopictus (26.4 (25.4-27.6); [47]). Together, these studies support the role of microscale variation in temperature as shaping the species segregation patterns in urban landscapes, and point towards a potential link of these segregation patterns to arboviral infection risk, which should be explored further.
We found few instances of coexistence between the species at the container scale (~9% of samples, Appendix B). The most frequent location for coexistence was in St. Vincent de Paul cemetery, which was a high heat cemetery dominated by Ae. aegypti. This cemetery is located near multiple water bodies that may promote a temperature mitigation effect in its surroundings that was not assessed by this study. More studies are needed to determine the factors that allow Ae. albopictus to inhabit this locality in spite of the high heat, and the potential role of proximity to water bodies as a mediating factor. Other instances where coexistence was detected at the vase level, shared the commonality of being closed canopy vases, and having Ae. albopictus at a higher relative abundance (Appendix B). These localities may be the focus of future studies to better assess the factors at the container scale that allow for coexistence, such as nutrient availability, and source and sink habitats.
The most abundant species collected in this study was Cx. quinquefasciatus, which was segregated temporally from the two Aedes species in cemeteries. A previous study in Tampa, Florida found a similar pattern, where Cx. quinquefasciatus switched habitat preference from cemeteries in the non-peak season, into other developed areas in the peak season, when the two Aedes species became more dominant [48]. The authors of the study suggest that coexistence among these competitors is enabled by spatial and seasonal segregation, and we provide further support for this finding in our study. We know from previous studies [49,50] and mosquito adult sampling in the city during 2020 (de Jesus, unpublished data) that Cx. quinquefasciatus is still abundant during the peak mosquito season in New Orleans. Therefore, the observed trend potentially reflects habitat switching to avoid competition with Ae. albopictus, which has been shown to be competitively superior to Cx. quinquefasciatus and other similar species such as Culex coronator, by laboratory studies testing interspecific competition under various resource levels [51,52].
Our study had several limitations. First, our monthly sampling was limited to seven cemeteries and one year of data. Longer-term studies at a larger scale would be necessary to fully understand spatiotemporal patterns of the mosquito species under study. Second, the temporal patterns reported on this study may have been affected by our sampling technique, which consisted of destructively sampling cemetery vases. This sampling strategy may have had an interference with the community assemblages in subsequent sampling events. However, since we limited our sampling to two cemetery vases per sampling occasion, while more were vases detected with water and larvae during our sampling, we think that our sampling interference was minimal. However, future studies may use non-destructive sampling strategies, such as oviposition traps to confirm our findings. Lastly, our sampling was limited to mosquito larvae, and we did not limit our collection to fourth instar larvae. Therefore, our survey does not fully reflect the outcomes of container scale competition or adult mosquito emergence, and more studies are needed to fully characterize the adult mosquito community assemblages across the city.

Conclusions
Ae. aegypti, and Ae. albopictus larvae show a pattern of spatial segregation in cemeteries of New Orleans. Ae. aegypti resides in high heat, low vegetation cemeteries, while Ae. albopictus was more often found in cemeteries with a lower heat index. Canopy cover over the container habitat in this highly urban environment is not predictive of Ae. albopictus, but it is predictive for Ae. aegypti. Ae. aegypti prefers to be under a shaded canopy in the high heat cemeteries where it resides. The qualities at the cemetery scale that are predictive of the habitat segregation patterns observed on this study (e.g., UHI) may be associated with dispersal limitation of Ae. albopictus under extreme heat conditions, but this hypothesis requires further evaluation. Our study suggests that high heat and low vegetation cover is characteristic of low-income neighborhoods in New Orleans, possibly highlighting multiple pathways for public health disparities in the city, including greater exposure to Ae. aegypti, which should be explored further. Data Availability Statement: Data generated in this study are available upon request.

Acknowledgments:
We would like to thank Emily Ford for facilitating access to cemeteries for sampling mosquito larvae, and Pete Aniello for providing access to the Urban Heat Island GIS layer used in this study. We acknowledge the field assistance of Zachary Taylor, Matthew Mount, and Hannah Orff. Donald Yee provided comments and suggestions to improve a previous version of this manuscript.

Conflicts of Interest:
The authors declare no conflict of interest.